Hydrogen (H2) is becoming an attractive alternative energy source to fossil fuels due to its clean emissions and potential for cost effective production by microorganisms. As such, microorganisms that metabolize H2 are being investigated for their potential use in H2-production. A microorganism of particular interest for H2 production is the green alga, Chlaydomonas reinhardtii, which is able to catalyze light-dependent, H2 production utilizing water as a reductant. Ghiradi et al., (2000) Trends Biotech. 18(12):506-511; Melis et al., (2001) Plant Physiol. 127:740-748. The benefits of using an algal system for H2-production include the use of renewable substrates (light and water) and its potential cost-effectiveness. Melis A, Int. J. Hyd. Energy 27:1217-1228. As such, there is a great deal of interest in optimizing H2-production by green algae to maximize the potential benefit as an alternative energy source.
Chlamydomonas reinhardtii, and other like microorganisms, are able to express a class of H2 metabolizing enzymes called hydrogenases. Members of this enzyme family function in either H2-uptake (as a means to provide reductant for substrate oxidation) or H2-production (as a means to eliminate excess reducing equivalents). Characterization of various hydrogenases from multiple organisms has identified three principle hydrogenase types, broadly classified by the chemical nature of their active sites: [Fe]-hydrogenase, [NiFe]-hydrogenase, and non-metallic (organic) hydrogenase. Vignais et al, (2001) FEMS Micro. Rev. 25:455-501; Adams. M. W., Biochem. Biophys. Acta. 1020:115-145; Buurman et al., (2000) FEBS Letts. 485:200-204. More particularly, [Fe]-hyrdogenase have an active site containing a [4Fe-4S]-center bridged to a [2Fe-2S]-center (H-cluster) (Peters et al., (1998) Science 282:1853-1858; Nicolet et al., (1998) Structure 7:13-23), and the [NiFe]-hydrogenase have an active site containing a [4Fe-4S]-center bridged to a [NiFe]-center (Volbeda et al., (1995) Nature 373:580-587). Coordination of the metal prosthetic groups to the active sites is made by cysteinyl, CN−, and CO ligands. Further, within each hydrogenase group are monomeric, or multimeric enzymes, that can be either cytoplasmic or membrane bound within the cell. Vignais et al., Supra.
Although there are differences within the active sites between different families of hydrogenase, as well as between the subunit composition and localization between hydrogenase families, most, if not all studied hydrogenases have exhibited some degree of sensitivity to inhibition by CO and O2. Adams M. W. W; Volbeda et al., (1990) Int. J. Hyd. Energy 27:1449-1461. Hydrogenase sensitivity to these inhibitors correlates to some degree to the type of prosthetic group that forms the active site, for example, [Fe]-hydrogenase is highly sensitive to O2. As such, for example, the activity of [Fe]-hydrogenase in C. reinhardtii is very sensitive to O2 during H2-photoproduction under photosynthetic conditions. Ghirardi et al., (1997) App. Biochem. Biotech. 63-65:141-151. Oxygen inhibition of [Fe]-hydrogenases is a major drawback in the use of green alga for H2 production.
One approach to overcoming this H2 production limitation is to stress the C. reinhardtii under photoheterotrophic, sulfur-deprived conditions that minimize O2-photoproduction levels and result in sustained H2-production. However, this approach does not result in optimal yields and requires the use of sulfur-deprived/oxygen limited production techniques. Recently, CO and O2 inhibition of hydrogenase activity in alga has been focused on the putative role of the H2-channel. For example, it has been shown that the positioning of the Fe2-atom in the enzyme's active site is directly at the active-site/H2-channel interphase, where it is easily accessed by either CO or O2 diffusing through the channel. Lemon et al., (1999) Biochem. 38:12969-12973; Bennett et al., (2000) Biochem. 39:7455-7460. Further, a naturally occurring O2-resistant [NiFe]-hydrogenase has been shown to have a narrower active site/H2-channel interphase than the naturally occurring hydrogenase counterpart. Volbeda et al. (2002), Supra.
Against this backdrop the present invention has been developed.